Abstract In this revised application, which focuses on bio-behavioral inflexibility as a framework to identify risk factors for juvenile-onset depression (JOD), we responded to each of the IRG's concerns. In particular, we: a) offer more explicit formulations of our model, hypotheses, and statistical approaches, b) address possible environmental moderators, and c) present the results of new pilot studies, which show our ability to induce and remediate dysphoric mood in Hungarian subjects and support the use of somatic probes of cardiac vagal control in youths. In this application, we propose to study the relation between current and prospective JOD risk and two functionally important bio-behavioral systems: cardiac vagal control (CVC) and mood repair. To characterize CVC inflexibility, we will assess CVC functioning across different experimental challenges (somatic vs. psychological); within versions of one type of challenge (e.g., alternative psychological tasks); and across different facets of CVC (resting vs. CVC reactivity and recovery). To characterize inflexible mood repair, we will assess subjects' ability to attenuate dysphoric mood subsequent to negative mood challenges by implementing 2 different mood repair strategies (positive autobiographical recall vs. distraction) and across different mood repair opportunities (experimentally controlled vs. unstructured). Our sample (11-18 years old at entry) will include 200 probands with JOD, 200 of their at-risk siblings not yet affected by JOD, and 100 never-ill controls. Probands (and siblings) will be recruited from a carefully diagnosed and well-characterized sample of 700+ young patients with JOD (each with at least one sibling), representing a national, clinical sample in Hungary, who participated in a recent Program Project. The design includes cross-sectional assessment of CVC, mood repair, psychiatric status, and psychosocial functioning, and longitudinal clinical follow-up. We propose that: (1) physiological inflexibility (operationalized as indices of impaired CVC) and behavioral inflexibility (operationalized as indices of impaired mood repair), and their combinations (global bio- behavioral inflexibility indices), will cross-sectionally distinguish probands, at-risk siblings, and control peers and that (2) global bio-behavioral inflexibility indices will prospectively predict clinical course (elevated depressive symptoms, recurrent depressive episodes in JOD probands, onset of first depressive episode in previously unaffected siblings). We also will test elements of our global inflexibility models in the presence of environmental moderators. Our study is significant because depression is a leading cause of disability worldwide. Moreover, JOD is a severe and recurrent form of depression that accounts for about 50% of the cases of depression in young adults. Our study is innovative because it: (a) integrates biological and behavioral indices relevant to depression that have ready translational potential, (b) uses alternate probes of CVC and mood repair across multiple stimulus conditions to obtain a richer characterization of these constructs, and (c) its bio-behavioral targets can inform improved efforts to detect, prevent, and treat depression.